The T7 RNA polymerase is a single-subunit enzyme that synthesizes RNA at a rate five times faster than E. coli RNA polymerase. It initiates transcription exclusively at the t7 promoter, a 23 bp sequence not found in the host genome.
The T7 bacteriophage is a prototype of the Podoviridae family, characterized by a non-contractile short tail and an icosahedral head. Unlike filamentous phages like M13, T7 is a lytic bacteriophage containing a linear double-stranded DNA (dsDNA phage) genome. It is renowned for its highly efficient gene expression machinery and robust stability, making it a powerful tool in modern biotechnology. This overview details the biology of the t7 bacteriophage, its application as a t7 expression system, and the utility of the t7 phage display system for protein engineering and interaction studies.
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T7 belongs to the class Caudoviricetes and the family Autographiviridae (formerly Podoviridae). Its structure is relatively simple yet extremely stable, capable of withstanding harsh conditions that would inactivate other viral vectors.
Fig.1
T7 bacteriophage structure.1
One of the most significant contributions of T7 biology to science is the development of the t7 expression system. This system relies on the exquisite specificity of the T7 RNA polymerase for its cognate promoter sequence.
The T7 RNA polymerase is a single-subunit enzyme that synthesizes RNA at a rate five times faster than E. coli RNA polymerase. It initiates transcription exclusively at the t7 promoter, a 23 bp sequence not found in the host genome.
Unlike filamentous phages which require secretion across the membrane, T7 assembly occurs in the cytoplasm. This allows for the cytoplasmic expression of proteins that are toxic to the secretion machinery or that fold rapidly in the reducing environment of the cytosol.
This system allows for the high-level production of recombinant proteins, often accumulating up to 50% of the total cellular protein. The t7 dna polymerase (gene 5 product) is another critical enzyme derived from this phage, widely used in DNA sequencing and replication studies due to its high processivity.
The t7 phage display system exploits the properties of the T7 lytic cycle and capsid structure to display peptides and proteins. This system, often referred to as the t7 select system, offers distinct advantages over M13-based systems, particularly for specific types of library construction.
In the T7 system, the exogenous DNA sequence is fused to the C-terminus of the 10B capsid protein gene. The 10B protein is a minor coat protein naturally present on the capsid surface. By fusing peptides to 10B, researchers achieve capsid display where the fusion proteins project outward from the phage head, accessible for binding interactions.
A unique feature of the T7 system is the ability to modulate display density. While M13 usually displays 1-5 copies, T7 can achieve high copy number display, presenting up to 415 copies of small peptides per phage particle. This high avidity makes it exceptionally sensitive for detecting low-affinity interactions.
Because T7 is a lytic phage that assembles in the cytoplasm, displayed proteins do not need to pass through the bacterial membrane. This is a critical advantage for cDNA library construction.
Recommended Service: T7 Phage Display System Construction
Our advanced T7 system offers a robust platform for displaying peptides and proteins. Unlike M13, the T7 phage assembles in the cytoplasm and is released by lysis, making it ideal for displaying cytoplasmic proteins and constructing unbiased cDNA libraries without secretion limitations. Leverage our expert platform for high-density peptide display and rapid biopanning.
The versatility of T7 phage makes it a preferred vehicle for diverse applications, ranging from epitope mapping to the discovery of novel protein interactions.
Our facility handles the amplification and purification of high-titer T7 phage stocks. We ensure high infectivity and purity for downstream applications like library screening or structural analysis.
Leverage the power of T7 for unbiased cDNA library screening. We construct custom libraries from specific tissues or cells to identify novel biomarkers, antigens, or protein interaction partners.
We also provide a broad range of related phage technologies. Explore our capabilities in M13 Phage Display System Construction for antibody engineering, or λ Phage Display System Construction for specific large-genome applications. For specialized needs, we offer Hyperphage Display System Construction and Phagemid and Helper Phage Dual-genome Display System Construction.
In a recent study, researchers developed a high-diversity T7 phage display sdAb library to identify novel ligands targeting chicken dendritic cells (DCs). DCs are crucial antigen-presenting cells for initiating adaptive immune responses, yet DC-targeting vaccines for poultry are underexplored. The team constructed a VHH library by inserting alpaca VHH genes into the T7 select 415-1b vector, achieving a library titer of 1.65 × 1011 PFU/mL with high diversity. Using intact chicken bone marrow-derived DCs as targets, they performed three rounds of bio-panning. This process successfully enriched for DC-specific binders, identifying 46 unique phage clones from 125 sequenced candidates. Notably, clones such as Phage-54 and Phage-74 demonstrated specific binding not only to chicken DCs but also to duck and goose DCs, without cross-reacting with fibroblasts. Confocal microscopy confirmed that these sdAb-displaying phages efficiently adsorbed onto the DC surface within 15 minutes, facilitating rapid internalization. Furthermore, immunization of specific-pathogen-free chickens with these phage clones elicited significantly higher antibody titers against the phage capsid compared to wild-type T7 phage. These findings underscore the utility of the T7 system for displaying functional sdAbs and its potential in developing targeted vaccine delivery vehicles for avian species.
Fig.2
T7 phage display sdAb library construction and bio-panning.2
Q: What is the main difference between T7 and M13 phage display?
Q: Can T7 phage display large proteins?
A: Yes, T7 can display relatively large proteins (up to ~1200 amino acids) as fusions to the capsid protein. However, for very large proteins, the copy number is typically reduced (0.1-1 copy per phage) to maintain capsid stability, whereas small peptides can be displayed in high copy numbers (up to 415).
Q: Is the T7 system suitable for antibody discovery?
A: While M13 is the standard for antibody (scFv/Fab) display due to the disulfide bond formation requirements in the periplasm, T7 can be used for antibody display if the antibodies are stable in the cytoplasm or if specific strains/conditions are used. T7 is more commonly used for peptide, cDNA, and scaffold protein libraries.
Q: Do I need a helper phage for the T7 system?
A: No, unlike phagemid-based M13 systems which require a helper phage for packaging, the T7 system typically uses a phage vector that contains all necessary genes for replication and assembly. The display is achieved by direct fusion to the capsid gene within the phage genome.
Q: What is the stability of T7 phage libraries?
A: T7 phage particles are extremely robust. They are stable across a wide range of pH and ionic conditions and can withstand harsh denaturing agents like urea or guanidine hydrochloride, making them ideal for biopanning under stringent conditions to select high-affinity binders.
Q: How many copies of a peptide can be displayed on T7?
A: The T7 Select system offers different vectors for varying display densities. The high-copy vectors (e.g., T7Select10-3) can display up to 415 copies of a peptide per phage particle (on every 10B capsid protein), providing high avidity for detecting weak interactions.
Q: Why is T7 preferred for cDNA libraries?
A: Since T7 assembles in the cytoplasm, it avoids the secretion bottleneck of the M13 system. This means that cytoplasmic proteins, which might fold prematurely or be toxic to the secretion machinery, are displayed efficiently. This allows for a more comprehensive representation of the proteome in cDNA libraries.
Q: How fast is the T7 amplification process?
A: The T7 lytic cycle is very rapid, typically completing within 20-30 minutes at 37°C. This allows for the amplification of libraries and multiple rounds of biopanning to be performed much faster than with filamentous phages, significantly accelerating the discovery timeline.
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